The present invention relates to devices and processes for determining concentrations of analytes in liquid solutions, and more particularly to the use of such apparatus and processes in combination with high performance liquid chromatography and other analytical separation methods.
A variety of separation methods are known for analyzing solutes in liquid media, including liquid chromatography, high performance liquid chromatography (HPLC), gel permeation chromatography capillary electrophoresis, centrifugation, and field flow fractionation. In all of these methods, it is essential to determine concentrations of analytes in the solution under study. Further, the ability to track changes in concentrations over time, corresponding to different regions or locations within a solvent eluting from a separation column, plays a key role in identifying the solutes involved. More particularly, these analytical systems typically employ detectors capable of generating a signal that varies with analyte concentration, yielding a chromatogram or plot of concentration verses time. Because different analytes (solutes) tend to travel through the separation column at different rates as the solution passes therethrough, different solutes exit the separation column at different times. Accordingly, regions of relatively high concentrations, temporally separated on the chromatogram, indicate the presence of several different solutes. In addition, each such region on the chromatogram corresponds to a region within the solution, in terms of differences in the time each such region exits the separation column. Such exit times are useful in identifying the solutes involved.
Generally, the detectors used in analytical separation systems are of two types: Selective detectors and universal detectors. Selective detectors respond only to a specific analyte or type of analyte. For instance, an ultraviolet absorbance detector responds only to molecules capable of absorbing ultraviolet light, e.g. proteins. An example of a universal detector is a refractive index detector, which responds to any analyte capable of changing the refractive index of the liquid that contains it.
One type of universal detector, introduced relatively recently but rapidly gaining acceptance for HPLC applications, is known as the evaporative light scattering detector (ELSD). This type of detector includes a nebulizer receiving a solution eluting from a separation column, then atomizing and spraying the solution as droplets, which dry to form residue aerosol particles. An air stream carries the residue particles past a beam of light, each particle scattering (reflecting or refracting) the light as it intersects the beam. One or more photodetectors sense the scattered light. The scattered light intensity increases with the size of the particle. Accordingly, the amplitude of the photodetector output signal is used to measure particle size.
Particle size is useful in determining concentration of the material forming the particle. If the nebulizer in the ELSD generates droplets at a constant size, the diameters of the resulting aerosol particles are proportional to the cube-root of the concentration. The intensity of scattered light is approximately proportional to the sixth power of the particle diameter for particles smaller than the wavelength of the coherent energy. Intensity is approximately proportional to the second power of the particle diameter for particles larger than that wavelength. The intensity/diameter relationship between these regions is complex. Thus, for small concentrations, the scattered light intensity is proportional to the square of the analyte concentration, while for high concentrations the scattered light intensity is proportional to concentration to the {fraction (2/3 )} power. With low concentrations being of primary interest in typical applications, the relationship of most concern is a variance of the output signal representing scattered light intensity as the square of the analyte concentration.
The ELSD is more sensitive than other universal detectors such as refractive-index detectors and viscosity detectors. Further, the ELSD responds to certain analyte molecules, such as polymers and carbohydrates, that do not provide a good ultraviolet or visible absorption signal. However, because of the square-law relationship just mentioned, the photomultiplier tube output signal rises rapidly with increasing concentration. As a result, the limited ranges over which photomultiplier tubes can operate impose severe limitations upon the dynamic range of the ELSD in terms of concentration measurements. ELSD systems can employ alternative detectors in lieu of photomultipliers. Nonetheless, the wide range of light intensities taxes the capabilities of these alternative detectors and the accompanying measuring circuitry. The practical dynamic range of an ELSD, in terms of a ratio of the highest measurable concentration to the lowest measurable concentration, is about 500.
A further problem associated with evaporative light scattering detectors is that the detector response is determined in part by the optical properties of the residue particles. In many cases these properties are largely unknown, requiring calibrations for each analyte under study.
In connection with analytical separation methods such as high performance liquid chromatography, but also more generally in the analysis of solutions as to the solutes they contain and their respective concentrations, it is an object of the present invention to provide a detector with a sensitivity at least comparable to the ELSD, which overcomes the aforementioned difficulties of the ELSD.
Another object is to provide a non-volatile analyte concentration detector with an enhanced dynamic range.
A further object is to provide a detector for determining analyte concentrations, with an output that varies with analyte concentrations according to a simpler relationship.
Another object is to provide a more compact instrument for measuring non-volatile analyte concentrations.
Yet another object is to provide a process for detecting concentrations of non-volatile analytes, for providing concentration measurements unaffected by the optical properties of the analyte particles involved.
To achieve these and other objects, there is provided a non-volatile analyte concentration detector. The detector includes an enclosure that defines a chamber. A first fluid passage is disposed to receive an aerosol stream composed of liquid droplets containing non-volatile material and suspended in a carrier gas. The first fluid passage is adapted to guide the aerosol stream toward the chamber as the liquid droplets substantially evaporate. As a result the aerosol stream as it enters the chamber is composed of reside particles of the non-volatile material. An ion generator is disposed near the chamber and adapted to generate multiple ions. A second fluid passage guides a gas flow toward the chamber and past the ion generator. The gas flow entrains at least a portion of the ions and carries the entrained ions into the chamber to merge with the aerosol stream, thus to apply a size-dependant electrical charge to each of the residue particles. The first and second fluid passages include respective first and second restrictions near the chamber to accelerate the aerosol stream and ion-carrying gas flow as they enter the chamber. A charge-responsive device is disposed downstream of an exit of the chamber to receive at least a portion of the charged residue particles. The device is adapted to generate an electrical signal having a level proportional to an aggregate charge of the received reside particles. Thus, the electrical signal indicates a concentration of the non-volatile material.
The charge-responsive device can include an electrically conductive filter adapted to entrap the reside particles, and a wire or other suitable electrical conductor electrically coupled with the filter. The level of electrical current through the conductor provides the indication of the non-volatile material concentration. Preferably the current is measured continuously, to provide a record of electrical current verses time over at least one selected time span corresponding to a selected sequence of the received residue particles. The electrical current level changes in response to changes in analyte concentration. More particularly, when the analyte concentration increases, each of the liquid droplets in the aerosol stream contains more of the non-volatile material. The residue particles that result from drying the aerosol are larger. The larger particles, when the aerosol stream merges with the ions, retain larger levels of electrical charge. The result is a more rapid accumulation of electrical charge at the filter, and a higher level of current in the conductor.
In theory, the level of charging in each particle is proportional to the particle diameter over a wide range of diameters. With concentrations being proportional to volumes as noted above, the particle charge levels, and thus the resulting conductor currents, should vary in proportion to the cube-root of the analyte concentration. Accordingly, a range in particle diameters encompassing two orders of magnitude provides a dynamic range encompassing six orders of magnitude, i.e. a factor of one million, for analyte concentrations.
In actual practice, based on a solution of sucrose in water, the detector electrical current has been found to vary more closely in proportion to the square-root of the concentration rather than the cube-root. This may be caused by coagulation in the aerosol, effects of analyte concentration on nebulizer performance, or other factors presently unknown. The resulting dynamic range, while not matching theoretical expectations, is a considerable improvement over the range afforded by the ELSD.
In a particularly useful application involving evaporative and electrical components, the analyte concentration detector is coupled to a nebulizer, with the first fluid passage receiving the nebulizer output. Either a pneumatic nebulizer or an electrostatic nebulizer may be employed. In the case of the electrostatic nebulizer, the aerosol leaving the nebulizer is neutralized before it is provided to the chamber for merger with the ions.
Another application of the invention, which presently is expected to gain widespread acceptance, is a high performance liquid chromatography system, in which the nebulizer receives a liquid sample from a liquid chromatography column or other separator. Upstream in the liquid chromatography system, a carrier liquid is provided to the liquid chromatography column at a substantially constant flow rate, with predetermined amounts of the liquid sample injected sequentially into the carrier liquid stream. As the liquid sample progresses through the separator, different non-volatile constituents travel through the separator at respective different rates. This tends to separate the liquid sample into regions corresponding to concentrations of the different non-volatile constituents. Alternatively, when a single non-volatile constituent is involved, different regions are characterized by different concentrations of the constituent.
When the liquid sample contains several different constituents which become concentrated within several different regions as the liquid exits the separator, a resulting record of electrical current verses time includes corresponding regions of constituent concentration, temporally separated from one another on the graph or other record to reflect the different times at which the different constituents exit the separator. Accordingly the record is useful not only for determining analyte concentrations, but also for identifying the analytes.
Further in accordance with the present invention, alternative systems can employ a variety of separators other than liquid chromatography columns. According to one alternative, field-flow fractionation, samples are injected into a flowing liquid stream. Different constituents are separated, based on different rates of travel in a transverse direction relative to the flow. In another alternative, a centrifuge is used to separate constituents based on different densities or sedimentation rates. Samples can be drawn out serially or in batches after centrifuge runs. Under another alternative, capillary electrophoresis, constituents in samples can be separated based on a variety of properties including their mobility in the liquid, size, and isoelectric points.
In accordance with any of these alternative separation methods, the system includes an analyte separator adapted to separate different non-volatile analytes in a liquid sample by concentrating different analytes primarily into different regions within the sample. In systems employing a separator that requires batch handling (e.g. a centrifuge), each sample batch is provided to the nebulizer in a manner that preserves the distinction among separate regions of the sample. Alternatively, separate regions of a sample batch can be provided to separate nebulizers.
A closely related aspect of the present invention is a process for measuring concentrations of non-volatile constituents in liquids, including:
a. providing a sample, including a liquid and at least one non-volatile constituent contained in the liquid, to a separator adapted to separate different non-volatile constituents from one another by concentrating different non-volatile constituents primarily into different regions within the sample;
b. receiving at least a portion of the sample exiting the separator, and using said portion to generate an aerosol stream composed of droplets including the liquid and the at least one non-volatile constituent;
c. allowing the liquid droplets to substantially evaporate, whereby the aerosol stream after evaporation is composed of residue particles of the at least one non-volatile constituent;
d. applying an electrical charge to each of the residual particles dependent on the residual particle""s size;
e. generating an electrical signal having a level proportional to an aggregate electrical charge of a selected sequence of the electrically charged residue particles in the aerosol stream; and
f. using the electrical signal to indicate a concentration of the at least one non-volatile constituent in the liquid sample over a region thereof corresponding to the selected sequence of the particles.
Thus in accordance with the present invention, solutions can be analyzed with respect to the concentrations of solutes with an accuracy comparable with that afforded by an evaporative light scattering detector, while avoiding the disadvantages associated with the ELSD. The use of electrical charge levels rather than scattered light intensity results in a considerably larger dynamic range over which concentrations can be determined with accuracy. The measurement of electrical charge (i.e. electrical current) rather than scattered light intensity also insures that concentration readings are not influenced by optical properties of the aerosol particles. For high performance liquid chromatography and a variety of other applications, the resulting concentration measurements are more reliable and can vary over a larger dynamic range.